Abstract

Interstitial cells of Cajal (ICC) regulate smooth muscle excitability and motility in the gastrointestinal (GI) tract. ICC in the deep muscular plexus (ICC-DMP) of the small intestine are aligned closely with varicosities of enteric motor neurons and thought to transduce neural responses. ICC-DMP generate Ca2+ transients that activate Ca2+ activated Cl- channels and generate electrophysiological responses. We tested the hypothesis that excitatory neurotransmitters regulate Ca2+ transients in ICC-DMP as a means of regulating intestinal muscles. High-resolution confocal microscopy was used to image Ca2+ transients in ICC-DMP within murine small intestinal muscles with cell-specific expression of GCaMP3. Intrinsic nerves were stimulated by electrical field stimulation (EFS). ICC-DMP exhibited ongoing Ca2+ transients before stimuli were applied. EFS caused initial suppression of Ca2+ transients, followed by escape during sustained stimulation, and large increases in Ca2+ transients after cessation of stimulation. Basal Ca2+ activity and the excitatory phases of Ca2+ responses to EFS were inhibited by atropine and neurokinin 1 receptor (NK1) antagonists, but not by NK2 receptor antagonists. Exogenous ACh and substance P (SP) increased Ca2+ transients, atropine and NK1 antagonists decreased Ca2+ transients. Neurokinins appear to be released spontaneously (tonic excitation) in small intestinal muscles and are the dominant excitatory neurotransmitters. Subcellular regulation of Ca2+ release events in ICC-DMP may be a means by which excitatory neurotransmission organizes intestinal motility patterns.

Significance Statement

Interstitial cells of Cajal (ICC) are innervated by enteric motor neurons and thought to transduce neural responses in GI muscles. Ca2+ transients, due to Ca2+ release from Ca2+ intracellular stores, mediate electrophysiological events in ICC by activation of Ca2+-activated Cl- channels (CaCCs). Neural responses in ICC in the deep muscular plexus (ICC-DMP) of the small intestine were studied by confocal imaging of Ca2+ transients in these cells. Excitatory neural input was due to cholinergic and peptidergic neurotransmitters [acetylcholine (ACh) and neurokinins], as excitatory effects on Ca2+ transients were blocked by atropine and neurokinin receptor antagonists. Neurokinins are the dominant excitatory regulators of Ca2+ transients in ICC-DMP. ICC-DMP are innervated by enteric motor neurons and mediate significant excitatory responses in intestinal muscles.

Introduction

Muscles of the gastrointestinal (GI) tract are innervated by both excitatory and inhibitory enteric motor neurons (Furness, 2012), and motility patterns of the gut depend on the outputs of the enteric nervous system. Neural inputs are overlaid on the basal excitability of the smooth muscle cells (SMCs) that line the walls of GI organs. SMC excitability is determined by ionic conductances and Ca2+ sensitization mechanisms intrinsic to these cells but also by interstitial cells that are electrically coupled to SMCs. Together SMCs and interstitial cells, i.e., interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor α-immunopositive (PDGFRα+) cells (Komuro, 2006; Sanders and Ward, 2006; Iino et al., 2009; Blair et al., 2012; Baker et al., 2013), form an electrical syncytium, known as the SIP syncytium (Sanders et al., 2012). It is the integrated output of these cells that determines the basal excitability of GI smooth muscle tissues and ultimately the responses to enteric motor neurons and other higher order regulatory pathways (Sanders et al., 2014a).

Excitatory neurotransmission in the gut is mediated predominantly via cholinergic neurotransmitters and neurokinins. The tachykinin (TKs) family of peptides [substance P (SP), neurokinin A (NKA) and NKB] is expressed throughout the GI tract (Holzer and Holzer-Petsche, 2001; Cipriani et al., 2011; Mitsui, 2011; Steinhoff et al., 2014). SP, NKA, and NKB are preferentially mediated through the stimulation of neurokinin 1 receptor (NK1), NK2, and NK3 G protein-coupled receptors. Both NK1 and NK2 receptors mediate contractile effects in the gut. Smooth muscle electrical, and motor events induced by electrical field stimulation (EFS) can involve both NK1 and NK2 receptors. But functional evidence supports the involvement of the NK1 subtype in mediating nonadrenergic noncholinergic (NANC) contractions to EFS in the mouse small intestine (Iino et al., 2004; De Schepper et al., 2005).

In the present study, we investigated the hypothesis that a major mechanism by which enteric motor neurotransmitters regulate ICC is through modulation of Ca2+ release events. To test this hypothesis, we explored whether excitatory neural inputs to ICC-DMP are coupled to Ca2+ release and characterized the nature of the Ca2+ responses that constitute this transduction pathway for postjunctional excitatory transmission.

Materials and Methods

Animals

GCaMP3-floxed mice (B6.129S-Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J) and their wild-type siblings (C57BL/6) were acquired from The Jackson Laboratory and crossed with Kit-Cre mice (c-Kit+/Cre-ERT2), provided by Dr. Dieter Saur (Technical University Munich, Munich, Germany). Kit-Cre-GCaMP3 mice (both sexes) were injected with tamoxifen at six to eight weeks of age (2 mg for three consecutive days), as previously described (Baker et al., 2016) to activate Cre recombinase and GCaMP3 expression. 15 days after tamoxifen injection, Kit-Cre-GCaMP3 mice were anaesthetized by isoflurane inhalation (Baxter) and killed by cervical dislocation. All animals used for these experiments were handled in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno.

Tissue preparation

Segments of jejunum (2 cm in length) were removed from mice and bathed in Krebs-Ringer bicarbonate solution (KRB). Jejunal segments were opened along the mesenteric border and luminal contents were washed away with KRB. The mucosa and sub-mucosa layers were removed by sharp dissection, and the remaining tunica muscularis was pinned flat within a Sylgard coated dish.

All drugs were purchased from Tocris Bioscience and dissolved in the solvents recommended by the manufacturer to obtain stock solutions. Final concentrations used in experiments were obtained by dilution into KRB.

Jejunal ICC were dispersed from Kit+/copGFP mice as previously described (Zhu et al., 2009; Zhu et al., 2011). ICC were sorted and purified by FACS (FACSAria II; Becton-Dickinson) using an excitation laser (488 nm) and emission filter (530/30 nm). Sorting was performed using a 130-μm nozzle and a sheath pressure of 12 psi.

RNA was prepared from sorted ICC and dispersed jejunal cells of the tunica muscularis before sorting using an illustra RNAspin Mini RNA Isolation kit (GE Healthcare). The PCR primers used and their GenBank accession numbers are provided in Table 1. qPCR was performed using SYBR green chemistry on the 7500 HT Real-time PCR System (Applied Biosystems) and analyzed, as previously described (Baker et al., 2016). All datasets were normalized to the housekeeping gene Gapdh.

Calcium event analysis

Analysis of Ca2+ activity in ICC-DMP was performed, as described previously (Baker et al., 2016). Briefly, movies of Ca2+ activity in ICC-DMP were converted to a stack of TIFF (tagged image file format) images and imported into custom software (Volumetry G8c, GW Hennig) for analysis. Tissue movement was stabilized to ensure accurate measurement of Ca2+ transients from ICC-DMP. Whole cell ROIs were used to generate spatio-temporal (ST) maps of Ca2+ activity in individual ICC-DMP. ST maps were then imported as TIFF files into ImageJ (version 1.40, National Institutes of Health; http://rsbweb.nih.gov/ij) for post hoc quantification analysis of Ca2+ events.

Experimental design and statistical analysis

Ca2+ event frequency in ICC-DMP was expressed as the number of events fired per cell per second (s−1). Ca2+ event amplitude was expressed as ΔF/F0, the duration of Ca2+ events was expressed as full duration at half maximum amplitude (FDHM), and Ca2+ event spatial spread was expressed as μm of cell propagated per Ca2+ event. Unless otherwise stated, data are represented as mean ± SEM. Statistical analysis was performed using either a Student’s t test or with an ANOVA with a Dunnett post hoc test where appropriate. In all statistical analyses, p < 0.05 was taken as significant; p < 0.05 are represented by a single asterisk (*), p < 0.01 are represented by two asterisks (**), and p < 0.001 are represented by three asterisks (***). When describing data throughout the text, n refers to the number of animals used in that dataset while c refers to the numbers of cells used in that same dataset.

Results

ICC-DMP displayed intracellular Ca2+ transients that fired in a stochastic manner (Fig. 1), as reported previously (Baker et al., 2016). Ca2+ transients were generated at multiple sites along the length of individual ICC-DMP and were typically localized, demonstrating no mechanism for active or regenerative propagation of these events within individual cells or between cells and no extrinsic mechanism of entrainment, as has been previously suggested (Huizinga et al., 2014). Ca2+ transients in ICC-DMP exhibit a range of frequencies, amplitudes, durations and spatial spread (Baker et al., 2016). ICC are thought to be intermediaries in enteric neurotransmission, relaying signals from enteric neurons to smooth muscle cells, that are electrically coupled to ICC (Daniel et al., 1998; Daniel and Wang, 1999). Therefore, we investigated how Ca2+ transients are modulated by enteric neurons activated by EFS.

ICC-DMP Ca2+ transient responses to nerve stimulation. A, Time-lapse montage showing postjunctional Ca2+ responses to EFS (10 Hz; 0.5-ms duration; 5 s) on an ICC-DMP in situ. An image of the GCaMP3 signal in the cell is shown in the leftmost panel. Scale bar for all panels: 25 μm. A color-coded overlay and calibration scale was imported to depict fluorescence intensity (F/F0) and enhance visualization of Ca2+ sites. Low fluorescence areas are indicated in dark blue or black. High-intensity fluorescence areas are indicated in red and orange. The “pre stimulation” panel shows a summed image of Ca2+ activity within the cell for 5 s before the onset of EFS, Ca2+ firing sites are marked with red asterisks. Panels showing the summed Ca2+ activity for the initial 2 s of EFS, the final 3 s of EFS and 5-s post-EFS are also shown. B, Representative ST map of Ca2+ transients in ICC-DMP shown in A. EFS duration is indicated by the dashed white box. The firing activities of three sites highlighted on the ST map are plotted in C, and the timing of EFS is indicated by the dashed red box.

EFS (10 Hz, 0.5 ms for 5-s trains) caused two distinct Ca2+ responses: (1) an initial inhibitory phase; (2) an excitatory response that occurred largely after cessation of EFS (Movie 1). The initial inhibitory response at the onset of EFS lasted about ∼2 s. During this phase, Ca2+ transients in ICC-DMP ceased (Fig. 1A–C). In the final 3 s of EFS, Ca2+ transients escaped from inhibition leading to an excitatory response that persisted into the period after cessation of the stimulus (Fig. 1A). These effects are illustrated by an ST map and Ca2+ activity traces in Figure 1B,C. This example demonstrates that in the final 3 s of EFS and particularly in the 5 s after cessation of EFS, Ca2+ transients were increased relative to the control period, and firing sites within ICC-DMP increased their firing frequency. We also found that the initiation sites for Ca2+ transients varied temporally in response to EFS (Fig. 1B). These responses were mediated by neuronal inputs, as they were blocked by tetrodotoxin (TTX, 1 μM, data not shown). As above, after the onset of EFS, an inhibitory response phase occurred, but in subsequent experiments we concentrated on the excitatory aspects of the neural responses.

Movie 1.

ICC-DMP Ca2+ transient responses to enteric neuronal stimulation. Movie of intracellular Ca2+ transients in ICC-DMP labeled with the genetically encoded Ca2+ indicator GCaMP3 in response to EFS (10 Hz, for 5 s; real-time playback). The top left FOV shows typical elongated ICC-DMP using a 60× objective (original recordings). Note that Ca2+ transients fired in stochastic fashion the blue bit-masked cell. The right window shows Ca2+ transient particles thresholded (SNR >= 25 dB, to facilitate visualization of active signals) after differentiation (Δt = 0.5 s) and smoothing (Gaussian 1.0 SD, box size = 3.3 µm) as shown in the middle window. Scale bar in top left window is 15 μm and pertains to all windows. The blue overlay of ICC-DMP in the FOV (blue bit-masked cell) was used to construct an ST map of Ca2+-induced fluorescence intensity across the diameter of the cell, which better displays the firing and propagation of Ca2+ transients along the length of the cell in response to EFS (lower panel; EFS duration is indicated with the yellow box). The bottom panel shows active area of Ca2+ transients across the FOV (area of active particles). Note the caseation of Ca2+ transients in response to EFS and enhanced Ca2+ firing during post stimulus period. Scale bar in the lower ST map and bottom active area map: 50 μm.

The excitatory Ca2+ response to EFS was quantified during the final 3 s of EFS (Fig. 2A, blue dashed box) and in the 5 s immediately following EFS (post-EFS; Fig. 2A, green dashed box). In the pre-EFS period, the control frequency of Ca2+ transients was 1.04 ± 0.08 events s−1, and this was increased significantly during the final 3-s period of EFS to 1.8 ± 0.15 events s−1 (Fig. 2B, p < 0.0001, n = 23, c = 56). The frequency of Ca2+ transients in the post-EFS period was also significantly increased from control, firing on average at 2.1 ± 0.1 events s−1 (Fig. 2B, p < 0.0001, n = 23, c = 56). There was a significant increase in Ca2+ transient amplitude in the final 3 s of EFS from 0.8 ± 0.06-1.1 ± 0.05 ΔF/F0 (Fig. 2C, p < 0.05, n = 23, c = 56), although there was no significant increase in amplitude in the post-EFS period compared to control (Fig. 2C, p > 0.05, n = 23, c = 56). Ca2+ transient duration increased in the final 3 s of EFS from 193 ± 3.7 to 219.6 ± 7.9 ms (Fig. 2D, p < 0.01, n = 23, c = 56) and was also significantly increased in the post-EFS period, increasing to 222 ± 6.5 ms (Fig. 2D, p < 0.001, n = 23, c = 56). Ca2+ transient propagation spread was also increased in the final 3 s of EFS from 11 ± 0.6 to 15.4 ± 0.9 μm (Fig. 2E, p < 0.001, n = 23, c = 56) and was also significantly increased, as compared to control, in the post-EFS period, with Ca2+ transients propagating an average of 12.9 ± 0.6 μm (Fig. 2E, p < 0.05, n = 23, c = 56). The number of Ca2+ firing sites in ICC-DMP was decreased significantly during the final 3 s of EFS (p < 0.001) and during the post-EFS period (p < 0.001; Fig. 2F, n = 23, c = 56). This is likely a result of the increased propagation spread of Ca2+ transients during these periods, as shown in Figure 2E. As the frequency of Ca2+ transients increased and they propagated over longer distances, individual firing sites may summate to create the increase in propagation distances observed during the final seconds of EFS and post-EFS. This could lead to an apparent reduction in firing sites, as the underlying sites were masked by propagating Ca2+ waves. A small increase in Ca2+ transient propagation velocity, that did not reach significance, was also observed during the final 3 s of EFS and during the post-EFS period (p < 0.05; Fig. 2G, n = 23, c = 56).

When cholinergic and NK1 receptors were both antagonized by adding both atropine (1 μM) and RP 67580 (1 μM), pronounced inhibition of Ca2+ transients persisted during the final 3 s of EFS and during the post stimulus period, as shown in Figure 10A–F (n = 4, c = 20).

Next, we inhibited cholinergic and neurokinin transmission with atropine and RP 67580 in the presence of L-NNA and MRS 2500. Under these conditions all Ca2+ transients were significantly diminished across all parameters tabulated, as shown in Figure 13A–F (n = 5, c = 32).

Discussion

Innervation of GI muscles by enteric motor nerves and the integrated firing of these neurons is essential for generating archetypal motility patterns (Spencer et al., 2016). ICC are innervated by enteric motor neurons, and their responses to neurotransmitters contribute to complex postjunctional responses of the SIP syncytium (Ward et al., 2000; Iino et al., 2004). In the case of the small intestine, ICC-DMP are an intramuscular type of ICC that are closely associated with and innervated by motor neurons (Rumessen et al., 1992; Zhou and Komuro, 1992; Wang et al., 2003b; Iino et al., 2004; Ward et al., 2006). We recently described the properties of spontaneous Ca2+ transients that occur in the absence of extrinsic stimuli in these cells (Baker et al., 2016). In the present study we investigated the effects of excitatory enteric motor neurotransmission on Ca2+ transients in ICC-DMP, because these events mediate activation of CaCC, the ion channels responsible for the electrophysiological postjunctional excitatory responses to nerve stimulation in small intestinal muscles (Zhu et al., 2011). EFS of intrinsic neurons resulted in three-component effects on Ca2+ transients: a brief inhibitory period (∼2 s), a period of escape from inhibition during sustained EFS, and a period of strong excitation after cessation of the stimuli (poststimulus or “rebound” excitation). The complexity of these responses is likely due to the fact that the enteric nervous system contains both inhibitory and excitatory motor neurons (Furness, 2012), and EFS can be expected to activate both classes of neurons.

In the mouse small intestine, the neurokinin component of the excitatory neural inputs to ICC-DMP was dominant. Our experiments also suggest that tonic release of neurokinins and binding to NK1 receptors is responsible for significant drive in generating the Ca2+ transients observed under basal conditions in ICC-DMP (Baker et al., 2016). Thus, the Ca2+ transients observed in the absence of applied stimuli are not “spontaneous” and do not appear to be driven intrinsically within ICC-DMP. Excitatory neurotransmitters greatly increased Ca2+ transients in ICC-DMP, and this mechanism likely underlies a portion of the postjunctional electrophysiological response to excitatory neural regulation (Zhu et al., 2011, 2015).

This study demonstrates that ICC-DMP receive and transduce excitatory neural inputs in the small bowel. Previous studies predicted this finding from morphologic observations (Rumessen et al., 1992; Zhou and Komuro, 1992; Wang et al., 2003a; Iino et al., 2004; Faussone-Pellegrini, 2006; Shimizu et al., 2008) and by showing that cholinergic excitatory neural responses develop in phase with the development of ICC-DMP and blocking Kit receptors causes parallel loss of ICC and cholinergic neural responses (Ward et al., 2006). Excitatory neurotransmission caused PKCɛ translocation in ICC-DMP that was blocked by atropine (Wang et al., 2003b), demonstrating functional cholinergic innervation and muscarinic responses in these cells. ACh binding to M3 receptors can enhance Ca2+ release in ICC-DMP via generation of inositol 1,4,5-trisphosphate (IP3) which activates Ca2+ release from the endoplasmic reticulum (ER). All of the molecular components of this pathway are expressed in ICC, as shown by transcriptome analyses (Chen et al., 2007; Lee et al., 2017). Previous direct observation of ICC-DMP in situ has shown that Ca2+ transients are due to Ca2+ release from intracellular stores (e.g., ER), mediated, in part, by IP3R (Baker et al., 2016). Increasing Ca2+ release in ICC leads to activation of CaCC, and the inward current generated by thousands of ICC-DMP in whole muscles would provide a net depolarizing influence that would summate with slow wave depolarizations, increase the likelihood of action potentials being generated during the plateau phase of slow waves (i.e., period of peak depolarization), and enhance the amplitude of phasic contractions (Zhu et al., 2011).

While our observations suggest innervation and contributions from cholinergic nerves to postjunctional excitatory responses, our data also suggest that neurokinins are the dominant excitatory neurotransmitters affecting Ca2+ transients in ICC-DMP in the mouse small intestine. ICC-DMP are closely associated with SP containing nerve fibers, and ICC-DMP express NK1 receptors (Iino et al., 2004; Faussone-Pellegrini, 2006; Shimizu et al., 2008) which is consistent with our observation that excitatory transmission to ICC-DMP was mediated through NK1 receptors. Previous studies have shown that exposure of small intestinal muscles to SP or stimulation of motor neurons causes internalization of NK1 receptors in ICC (Lavin et al., 1998; Iino et al., 2004). Our experiments showed that two NK1 receptor antagonists greatly attenuated basal Ca2+ transients and suppressed responses of ICC-DMP to EFS. The strong inhibitory effects of NK1 antagonists on Ca2+ transients could possibly be due to off-target effects on Ca2+ stores or Ca2+ release mechanisms; however, nonspecific effects do not appear to be significant because responses to CCh on Ca2+ transients were intact in the presence of the NK1 antagonist, RP 67580. Taken together these findings support the importance of neurokinin signaling in shaping motility patterns in the small intestine.

The degree to which basal Ca2+ transients were affected by NK1 antagonists in the present study was somewhat surprising. These results suggest ongoing release of neurokinins (i.e., tonic excitation), similar in concept to the tonic inhibition phenomena observed in many GI muscles (Wood, 1972; Lyster et al., 1995). Although this phenomenon has not been described previously in the small intestine, tonic activation of NK1 receptors has been proposed in other systems (Henry et al., 1999; Jasmin et al., 2002). In the present study attenuation of Ca2+ transients by the NK1 receptor antagonists may be caused by continuous release of neurokinins or persistence of the ligand in the spaces between motor nerve varicosities and ICC-DMP.

The enhanced relative reliance on neurokinins for excitatory effects may be due, in part, to the high expression of NK1 receptors by ICC-DMP which does not appear to be true for intramuscular ICC in the colon (Lee et al., 2017). NK1 receptors also couple to cellular responses through activation of phospholipase C and generation of IP3 (Steinhoff et al., 2014). Thus, there is a signaling pathway available for the enhancement of Ca2+ transients in ICC-DMP. However, it should also be noted that transfection of neurokinin receptors in model cells has also been associated with activation of adenylate cyclase and production of cAMP (Steinhoff et al., 2014), a pathway not typically linked to enhanced release of Ca2+. Generation of cAMP and stimulation of cAMP-dependent protein kinase is known to enhance Ca2+ sequestration into stores by phosphorylation of phospholamban (highly expressed in ICC; Lee et al., 2017) and stimulation of SERCA (Stammers et al., 2015). Perhaps increased loading of Ca2+ stores contributes to augmentation of Ca2+ transient amplitude and spatial spread by neurokinins, and enhancing the rate of recovery of Ca2+ into stores after a release event, reducing the time required for a given store to generate another Ca2+ transient.

In summary, this study supports the idea that significant neural regulation occurs in the intramuscular class of ICC in the small intestine (ICC-DMP). As discussed above, much of the excitatory response was mediated through NK1 receptors that are expressed largely by ICC-DMP (Sternini et al., 1995; Vannucchi et al., 1997; Iino et al., 2004). Responses to EFS were attenuated by NK1 antagonists. Previous studies have shown that electrophysiological responses in ICC-DMP are linked to Ca2+ release events (Zhu et al., 2011; Zhu et al., 2015), suggesting that Ca2+ transients in ICC-DMP couple to generation of inward currents and depolarizing effects on the SIP syncytium. NK2 receptors, expressed largely by SMCs (Cipriani et al., 2011), were apparently not involved in responses of ICC-DMP to neurokinins released from nerve terminals, because an NK2 antagonist had no effect on responses. The effectiveness of neurokinins as neurotransmitters in the tunica muscularis of the small intestine may be spatially confined by concentrations achieved in postjunctional spaces to a subset of neurokinin receptors expressed by ICC-DMP.

Acknowledgments

Acknowledgements: We thank Ms. Nancy Horowitz for help with breeding of animals and tamoxifen treatments. ICC were purified by FACS from enzymatic dispersions of jejunal muscles in the Cell Cytometry and FACS Core Laboratory supported by a Phase III COBRE Award from the National Institute of General Medical Sciences Grant P30-GM110767. This Core was developed and supervised by the late Dr. Douglas Redelman and more recently by Mr. Byoung Koh.

Footnotes

The authors declare no competing financial interests.

This work was supported by Department of Health and Human Services National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases Grants P01 DK41315 and P30-GM110767.

GoyalRK (2016) CrossTalk opposing view: interstitial cells are not involved and physiologically important in neuromuscular transmission in the gut. J Physiol594:1511–1513.doi:10.1113/JP271587pmid:26842563

ZhouDS, KomuroT (1992) Interstitial cells associated with the deep muscular plexus of the guinea-pig small intestine, with special reference to the interstitial cells of Cajal. Cell Tissue Res268:205–216.pmid:1617694

Synthesis

Reviewing Editor: Vivian Budnik, University of Massachusetts

Decisions are customarily a result of the Reviewing Editor and the peer reviewers coming together and discussing their recommendations until a consensus is reached. When revisions are invited, a fact-based synthesis statement explaining their decision and outlining what is needed to prepare a revision will be listed below. The following reviewer(s) agreed to reveal their identity: Nick Spencer

the current manuscript addresses all the criticisms raised by reviewers.